IMPROVING THE ABILITY OF ASTRAGALUS TRIGONUS L. TO PRODUCE APIGENIN USING MODERN GENETIC TECHNIQUES

Abstract

Astragalus trigonus considered an important medical plant had been used as traditional medicine, which improve the resistance to viral infections, hepatoprotective, heart tonic, nephritis and diabetes. For the purpose of genetic conservation of the Astragalus trigonus, an efficient and repeatable in vitro propagation system from the hypocotyl explants was developed. Murashige and Skoog medium (MS) included various concentration and /or of a-naphthaleneacetic acid (NAA), benzylamino-purine (BAP), thidiazuron (TDZ) and kinetin (KIN) was used for shoot initiation. The number of shoot/explants was high (14 shoots) in the presence of 0.5 mg/l NAA and 0.5 mg/l BAP. Elongated shoots were successfully rooted in MS medium supplemented with 1.0 mg/l NAA. High similarity percentage was detected among the in vitro regenerated plant with the mother plants as reveled by the molecular marker analysis; random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR). The data obtained from both of RAPD and ISSR analyses showed 35.2 % polymorphism among the regenerated and their mother plants. The protocol in this study can be applied to conserve the endangered Astragalus trigonus. in order to enhancement the apigenin callus induction was achieved on MS supplemented with 0.5 mg/l 2,4-D and the elicitation with jasmonic acid, salicylic acid, chitosan and yeast extract was tested. Yeast extract in 100 mg/l scored the high Apigenin content mg/g dry weight approximately (1.96 mg/g) 2.56-fold of apigenin in mother plant. while transformation with Agrobacterium tumefaciens harboring Plant vector pFGC5941 / f3′5′h scored the highest percentage of Apigenin content mg/g dry weight (13.86 mg/g) equal 18-fold Apigenin content in mother plant. Bar gene selection, PCR, Dot blot and Reverse Transcription-PCR techniques confirmed the integration of the f3′5′h gene into transgenic Astragalus trigonus genome